1669 Original Article Page 1 of 15 Intestinal mRNA expression profile and bioinformatics analysis in a methamphetamine-induced mouse model of inflammatory bowel disease Jiaxue Sun1,2,3#, Fengrong Chen1,3#, Cheng Chen1,2,3, Zherui Zhang1,2,3, Zunyue Zhang1,3, Weiwei Tian1,3, Juehua Yu1,3^, Kunhua Wang1 1NHC Key Laboratory of Drug Addiction Medicine, First Affiliated Hospital of Kunming Medical University, Kunming, China; 2Department of Gastrointestinal and Hernia Surgery, First Affiliated Hospital of Kunming Medical University, Kunming, China; 3The Scientific Research Laboratory Center, First Affiliated Hospital of Kunming Medical University, Kunming, China Contributions: (I) Conception and design: J Sun, F Chen, J Yu; (II) Administrative support: J Yu, K Wang; (III) Provision of study materials or patients: C Chen, Z Zhang, Z Zhang, W Tian; (IV) Collection and assembly of data: C Chen, Z Zhang, Z Zhang, W Tian; (V) Data analysis and interpretation: J Sun, J Yu; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. #These authors contributed equally to this work. Correspondence to: Juehua Yu, PhD. The Scientific Research Laboratory Center, First Affiliated Hospital of Kunming Medical University, Yunnan, China. Email: [email protected]; Prof. Kunhua Wang. NHC Key Laboratory of Drug Addiction Medicine, First Affiliated Hospital of Kunming Medical University, Yunnan, China. Email: [email protected]. Background: Methamphetamine use has become a serious global public health problem and puts increasing burdens on healthcare services. Abdominal complications caused by methamphetamine use are uncommon and often go ignored by clinicians. The exact intestinal pathological alterations and transcriptomic responses associated with methamphetamine use are not well understood. This study sought to investigate the transcriptome in a methamphetamine-induced mouse model of inflammatory bowel disease (IBD) using next-generation RNA sequencing. Methods: Tissues from the ileum of methamphetamine-treated mice (n=5) and control mice (n=5) were dissected, processed and applied to RNA-sequencing. Bioinformatics and histopathological analysis were then performed. The expression profiles of intestinal tissue samples were analyzed and their expression profiles were integrated to obtain the differentially expressed genes and analyzed using bioinformatics. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the differentially expressed genes were performed using Metascape. Results: A total of 326 differentially expressed genes were identified; of these genes, 120 were upregulated and 206 were downregulated. The Gene Ontology analysis showed that the biological processes of the differentially expressed genes were focused primarily on the regulation of cellular catabolic processes, endocytosis, and autophagy. The main cellular components included the endoplasmic and endocytic vesicles, cytoskeleton, adherens junctions, focal adhesions, cell body, and lysosomes. Molecular functions included protein transferase, GTPase and proteinase activities, actin-binding, and protein-lipid complex binding. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the differentially expressed genes were mainly involved in bacterial invasion of epithelial cells, protein processing in the endoplasmic reticulum, regulation of the actin cytoskeleton, and T-cell receptor signaling pathways. A set of overlapping genes between IBD and methamphetamine-treated intestinal tissues was discovered. Conclusions: The present study is the first to analyze intestinal samples from methamphetamine-treated mice using high-throughput RNA sequencing. This study revealed key molecules that might be involved in ^ ORCID: 0000-0002-1661-0503. © Annals of Translational Medicine. All rights reserved. Ann Transl Med 2020;8(24):1669 | http://dx.doi.org/10.21037/atm-20-7741 Page 2 of 15 Sun et al. Intestinal mRNA profile in a methamphetamine mouse model the pathogenesis of a special type of methamphetamine-induced IBD. These results offer new insights into the relationship between methamphetamine abuse and IBD. Keywords: Methamphetamine; gastroenterology; transcriptomics Submitted Oct 23, 2020. Accepted for publication Dec 21, 2020. doi: 10.21037/atm-20-7741 View this article at: http://dx.doi.org/10.21037/atm-20-7741 Introduction impaired innate immune function in methamphetamine- dependent rats, leading to inflammation and infection. Methamphetamine abuse has become a serious global Another cellular study found that methamphetamine public health concern. In 2018, over 1.9 million people can cause NLRP3 inflammasome overexpression and aged 12 years and above used methamphetamine in the severe intestinal inflammatory injury (12). Furthermore, United States, which equates to about 0.7% of the country’s inhibiting the expression of microRNA (miRNA) 181c, population (1,2). The China: 2019 Drug Situation Report which directly targets and regulates tumor necrosis factor-α, reported methamphetamine to be the most widely used has been shown to induce intestinal epithelial apoptosis, drug in China, with 1.186 million users. causing damage to the intestinal barrier (13). However, the The production and use of methamphetamine create mechanism of methamphetamine use impairs the immune the perfect conditions for adverse medical and social function and the intestinal barrier remains unclear. complications (3). Methamphetamine use results in To better understand the integrated mechanism of significant damage to almost all systems in the human body, immune function and intestinal barrier, we established a including the neurologic, cardiac, pulmonary, and dental mouse model to evaluate the effects of methamphetamine systems (3). This damage triggers a series of hyper-acute, use on pathological alterations and transcriptomic responses acute, and chronic complications, including the release in the gastrointestinal system, and demonstrated that of the neurotransmitters dopamine, norepinephrine, and methamphetamine causes a specific type of inflammatory serotonin, as well as activation of the cardiovascular and bowel disease (IBD). The results of this study provide a central nervous systems, vasoconstriction, pulmonary greater understanding of the molecular mechanisms in hypertension, atherosclerotic plaque formation, cardiac response to methamphetamine use in mice. arrhythmias, and cardiomyopathy (4-6). As a result, the We present the following article in accordance with the user’s immune system is significantly impacted, rendering ARRIVE reporting checklist (available at http://dx.doi. the body susceptible to infections and exacerbating the org/10.21037/atm-20-7741). severity of disease (7). Methamphetamine use is also associated with adverse psychological effects, such as euphoria, paranoia, agitation, mood disturbances, anxiety, Methods psychosis, depression, delusions, and violent behavior (3,8). Animals and drugs The release of dopamine and other neurotransmitters induced by methamphetamine can also have multiple Experiments were performed under a project license harmful effects on the gastrointestinal system. However, (2018NS0083) granted by Animal Care and Use abdominal complications due to methamphetamine Committee of Kunming Medical University, in compliance use are uncommon and often ignored by clinicians (9); with the National guidelines for the care and use of consequently, the resulting gastrointestinal pathology animals. Ten male specific-pathogen-free C57BL/6 mice has rarely been reported. Although some cases of (8-week, weight, 23.56–25.49 g) were supplied by the Jingda life-threatening abdominal complications caused by Laboratory Animal Co., Ltd. (cat. no. 1107271911003159). methamphetamine-induced mesenteric ischemia have been The mice were housed in ventilated cages with 5 animals described previously (3,9,10), systematically organized per cage and given ad libitum access to rodent chow and domestic studies are lacking. A previous study (11) reported water. All experiments were conducted during the light that gut dysbiosis impaired the intestinal barrier and period (between 07:00 a.m. and 07:00 p.m.) © Annals of Translational Medicine. All rights reserved. Ann Transl Med 2020;8(24):1669 | http://dx.doi.org/10.21037/atm-20-7741 Annals of Translational Medicine, Vol 8, No 24 December 2020 Page 3 of 15 The mice were divided into two groups and were cDNA library preparation and sequencing randomly allocated to receive either methamphetamine Sequencing libraries were generated using the NEB (treatment group, n=5) or saline vehicle solution (0.9% Next® UltraTM RNA Library Prep Kit for Illumina® sodium chloride; control group, n=5). Treatment (NEB#E7530, USA), following the manufacturer’s commenced when the mice reached 6 months old. The recommendations. Messenger RNA (mRNA) was purified treatments were administered in the mouse housing room using poly-T oligo-attached magnetic beads and then via intraperitoneal injection following an escalating dosing fragmented with divalent cations in reaction systems [NEB regimen to mimic patterns of drug abuse in humans Next First Strand Synthesis Reaction Buffer (5X)] under (Figure 1A). During the first week of treatment, mice in the elevated temperatures. Synthesis of first-strand cDNA treatment group were injected once daily with 2.5 mg/kg was performed using the random hexamer primers with of methamphetamine in the morning (between 8 and M-MuLV Reverse Transcriptase (RNase